Active agents which are preferably functional organic materials such as proteins, have been immobilized in the past on a wide variety of solid supports for various known applications including analysis, separation, synthesis and detection of biological and other materials. Often hydrophilic polymers have been used to immobilize the proteins because it is less difficult to attach proteins to polymers than to inorganic materials. However, there is an increasing need to immobilize functional organic material such as proteins on inorganic material such as silica, glass, silicon, metals and the like. Problems can arise with nonspecific protein adsorption to inorganic material.
Proteins such as antibodies can be attached to quartz or glass by simple adsorption, however, often such immobilized proteins can partially de nature and tend to leech or wash off the surface. Van der Merwe (U.S. Pat. No. 4,478,946) suggests the adsorption of nonfunctional proteins to a surface and the employment of crosslinking agents to covalently attach the functional proteins to the adsorbed nonfunctional proteins. However, the adsorption approach gives less than desirable attachment.
The prior art has suggested modifying surface hydroxyl groups of inorganic substrates to provide functionality that reacts directly with proteins (see U.S Pat. No. 4,415,665, U.S. Pat. No. 4,582,875 and U.S. Pat. No. 4,562,157). European patent No. 87,401,000.
Various methods for coating inorganic substrates with silane films have been reviewed, Weetal H. H. (1976) Methods in Enzymology, Volume 44, 134-148, Academic Press, New York, NY. Inorganic porous substrates coated with epoxy silane have been oxidized to produce aldyhyde groups reacting directly with antibodies, Sportsman, J. R. et al (1980) Anal. Chem. 52,2013-2018. Others, such as Sagiv U.S. Pat. No. 4,539,061, have established multilayers of silanes deposited on silica. Proteins have further been linked to silane films on silica using glutaraldehyde. See U.S. Pat. No. 4,478,946, Mandenius, C.F., et al (1984) Anal. Biochem. 137, 106-114, and Richards, F. M. et al (1968) J. Mol. Biol. 37, 231-233. Reactive crosslinkers such as glutaraldehyde may bind to many residues and form multi protein complexes which could interfere with protein function. To avoid the use of glutaraldehyde, others have modified silica surfaces with epoxy silanes and subsequently altered the silanes to have a dihydroxy terminus, U.S. Pat. No. 4,562,157. This method involved many steps and can have problems with the density of protein adsorbed to epoxy silane film. More recently, Jonsson, U. et al, Volume 137, 381-394 Methods in Enzymoloqy (1988) has suggested the use of silane films on specific inorganic surfaces and specific crosslinking agents which can be heterobifunctional, but in all cases, appear to require a free sulfhydryl group on the protein to be bound. This requires that nearly all proteins be modified prior to immobilization. Thus either a free-SH must be attached (a chemical modification which must be carefully controlled in order to avoid damage to the protein) or existing S-S bonds must be reduced. In the case of attaching antibodies to an amino terminal silane, this approach involves proteolysis and reduction to produce Fab fragments from intact antibodies. Such fragments can have reduced binding affinity for antigen and significant amounts can be lost during processing.